Development of eco-friendly spectrophotometric methods for analysis of metformin hydrochloride and linagliptin in presence of metformin toxic impurity in their pure and dosage forms: Validation, practicality and greenness studies.

Metformin is considered as type 2 diabetes first line treatment according to American Diabetes Association and European Association. But, in some cases, di- or tri - therapy should be prescribed for glycemic management, prevention of the maximum dose side effects and induced effectiveness. Co-administration of Linagliptin with metformin has many benefits on diabetic patients such as decrease the possibility of hypoglycemia. For the first time, novel and reliable techniques were developed and verified for the concurrent quantification of metformin hydrochloride and linagliptin, while accounting for the existence of metformin toxic impurity 1-cyanoguanidine in their pure and dosage forms. Method (A) utilizes the zero-order spectrophotometric approach to quantitatively determine the concentration of linagliptin. The measurements are performed at a wavelength of 295 nm. The double divisor derivative ratio spectrophotometric method is used in Method (B) to measure the amounts of metformin and cyanoguanidine at 252 nm and 219 nm wavelengths, respectively. The spectrophotometric method (C) for determining metformin and cyanoguanidine at 252 nm and 223 nm, respectively, is based on the single divisor derivative ratio-zero crossing technique. The obtained findings were subjected to statistical comparison with the reported method, revealing no statistically significant differences. The Green Analytical Procedure Index (GAPI) and Analytical GREEnness Metric approach (AGREE) determined that these approaches had a high degree of environmental friendliness. Additionally, the proposed strategy was deemed to be practical according to the Blue Applicability Grade Index (BAGI) assessment tool.

Innovative UV Protocols Based on Straightforward Mathematical Filtration for Concurrent Estimation of Two Antidiabetic Drugs in Their Brand-New Combination: A Comparative Study.

BACKGROUND: In 2019, the U.S. Food and Drug Administration approved a brand-new combination of linagliptin and empagliflozin in a formulation called Glyxambi® tablets for managing type 2 diabetes mellitus. Nowadays, spectrophotometric techniques occupy the first place among their peers in terms of ease of application, friendliness to the environment, and low costs. OBJECTIVE: This research discusses the development of two very simple spectrophotometric protocols based on zero-order spectra for the determination of linagliptin and empagliflozin. METHODS: The developed protocols were the induced dual-wavelength and absorption correction protocols. Linagliptin could be determined directly at 305 nm, at which the empagliflozin spectrum was zero-crossing. Empagliflozin was determined using the two developed protocols. The induced dual-wavelength technique was developed by calculating the equality factor of linagliptin to cancel its interference. The absorption correction technique was developed by measuring the correction absorption factor. RESULTS: The concentration ranges of linagliptin and empagliflozin were 1-10 µg/mL and 3-30 µg/mL, respectively. Excellent recovery results were found in bulk, dosage form, and synthetic mixtures. Low LOD and LOQ values were obtained, indicating the high sensitivity of the protocols. The statistical Student's t-test was performed to compare the results of the applied and reported protocols, indicating no difference between them. CONCLUSION: The proposed protocols have the advantages of being straightforward, affordable, and requiring no sophisticated manipulations, just simple mathematical calculations. The proposed protocols are acceptable for routine usage in QC laboratories and in future research applications. HIGHLIGHTS: Two novel univariate methods were developed for quantitative analysis of linagliptin and empagliflozin in their pharmaceutical and laboratory mixtures, and produced satisfactory results.

Application of experimental design in HPLC method optimization and robustness for the simultaneous determination of canagliflozin, empagliflozin, linagliptin, and metformin in tablet.

Gliflozins and gliptins represent two different pharmacological drug classes that exert different and potentially complementary glucose-lowering effect in patients with type II diabetes mellitus. A novel, selective, and sensitive HPLC method was developed for the determination of canagliflozin, empaglifozin, linagliptin, and metformin in pure form, in laboratory prepared mixtures, and in pharmaceutical dosage form. Experimental design optimization was applied by using Plackett-Burman and face-centered composite designs to achieve the best resolution with minimum experimental trials. Three significant variables affecting optimization, namely buffer pH, percentage of methanol, and percentage of acetonitrile, were studied. Chromatographic separation was achieved using an Agilent Eclipse C8 column, and column temperature was kept at 45°C. The mobile phase was composed of dipotassium hydrogen phosphate buffer (0.05 M, adjusted to pH 6 using o-phosphoric acid):acetonitrile:methanol (50:25:25, v/v/v) at a flow rate of 1.5 mL/min. Sharp and well-resolved peaks of the cited drugs were obtained. The method was fully validated in terms of linearity, accuracy, precision, selectivity and robustness in agreement with the International Council of Harmonization (ICH) guidelines Q2 (R1). Satisfactory results were obtained by the analysis of tablets through applying the developed method. Therefore, it could be performed for the analysis of the cited drugs in quality control laboratories.

Determination of linagliptin and empagliflozin by UPLC and HPTLC techniques aided by lean six sigma approach.

Two chromatographic techniques were developed and validated for simultaneous determination of the newly co-formulated antidiabetic combination linagliptin and empagliflozin in their pure form and film-coated tables. The first technique was UPLC; the separation and resolution of both analytes were achieved using a Zorbax eclipse plus C18 column applying an isocratic elution based on phosphate buffer pH 4-acetonitrile (65:35, v/v) as a running mobile phase at flow rate 1.5 ml/min and the effluent was monitored at 220 nm. Augmentation of Lean Six Sigma with UPLC and HPTLC methods had a major impact on the development of robust specifications to ensure that the quality at six sigma level has a high level of statistical confidence and target performance. On the chromatogram, empagliflozin and linagliptin appeared at retention times of 1.417 and 2.453 min, respectively. The second technique was HPTLC; both analytes were fairly well resolved and separated using a developing mobile phase composed of ethyl acetate-chloroform-acetonitrile (55:25:20 by volume). The values of retention factor (RF ) were 0.29 and 0.53 for linagliptin and empagliflozin, respectively. All variables were investigated to adjust the whole conditions.

Stability-indicating ultra performance liquid chromatography method development and validation for simultaneous estimation of metformin, linagliptin, and empagliflozin in bulk and pharmaceutical dosage form.

A rapid stability-indicating reversed phase-ultrapure liquid chromatography (RP-UPLC) was developed and validated for the estimation of metformin (MET), linagliptin (LIN), and empagliflozin (EMP) combination in bulk and tablet dosage form using Kromasil C18 column (2.1 × 50 mm, 1.8 µm) as a stationary phase and a mixture solution of 40% phosphate buffer (pH = 3) and 60% acetonitrile as mobile phase at a flow rate of 0.6 mL/min. The detection was performed at 248 nm using a photodiode array detector. The linearity, sensitivity, selectivity, robustness, specificity, precision, and accuracy were determined. The peak area response-concentration curve was rectilinear over the range of 50-150 (MET), 5-15 (LIN), and 10-30 µg/mL (EMP) with quantitation limits of 0.042 (MET), 0.023 (LIN), and 0.059 µg/mL (EMP). The proposed method was successfully validated for the determination of MET, LIN, and EMP simultaneously in combined tablet dosage form. The performance of the proposed method was compared with reported RP-UPLC methods and found to be rapid and economical. The developed and validated stability-indicating RP-UPLC method was appropriate for the quality control and drug analysis.

A capillary electrophoresis method for the determination of the linagliptin enantiomeric impurity.

Linagliptin is a highly specific, long-acting inhibitor that is used as an orally administrable agent for type-2 diabetes treatment. Because only the R-enantiomer is of clinical use, we developed a capillary electrophoresis method for the determination of the enantiomeric impurity of this compound. Carboxymethyl-ß-cyclodextrin was selected as the chiral selector for the separation of linagliptin enantiomers. Design of experiments and desirability functions were used for the analytical optimization, which was focused on understanding and improving the electrophoretic process. The effects of significant parameters (background electrolyte concentration and pH, cyclodextrin concentration, temperature, and voltage) were thoroughly investigated. The complete separation of linagliptin and its enantiomeric impurity with baseline resolution was achieved within 10 min on an uncoated fused-silica capillary (50 µm inner diameter, 365 µm outer diameter, 64.5/56 cm in total/ effective length) maintained at 25°C, under an applied voltage of 28.0 kV. The background electrolyte contained 70 mM sodium acetate and 4.7 mM carboxymethyl-ß-cyclodextrin, and the pH was adjusted to 6.10. The method was validated, and a limit of quantitation of 0.05% for the impurity was estimated.

Square-wave Adsorptive Anodic Stripping Voltammetric Determination of Antidiabetic Drug Linagliptin in Pharmaceutical Formulations and Biological Fluids Using a Pencil Graphite Electrode.

A simple, sensitive, low-cost, quick and reliable square-wave anodic stripping voltammetric method is described for the determination of the antidiabetic drug Linagliptin (LNG) in pure form, tablets, and spiked human urine and plasma samples. Using a pencil graphite electrode (PGE), cyclic voltammetry (CV) was applied to study the electrochemical behavior of LNG. In a Teorell-Stenhagen buffer (pH 5.5) containing 0.1 M NaClO4 as a supporting electrolyte, the LNG yields an irreversible well-defined oxidation peak at about 1.2 V vs. Ag/AgCl electrode. The various affecting factors, such as the pH, buffer type, supporting electrolyte, accumulation potential, scan rate and accumulation time, were tested and optimized. Also, square-wave adsorptive anodic stripping voltammetric (SWAdASV) studies show that the peak current various linearly over the LNG concentration range of 0.24 - 5.20 µg mL-1 (R2 = 0.9994). The detection and quantification limits were calculated to be 0.10 and 0.33 µg mL-1, respectively. The proposed procedure exhibits a good precision, selectivity, and stability and was applied successfully to determine the LNG in pharmaceutical formulations (tablets) and biological fluids (spiked human urine and plasma samples).

Mean centering-triple divisor and ratio derivative-zero crossing for simultaneous determination of some diabetes drugs in their quaternary mixture with severely overlapping spectra.

Two ratio derivative spectrophotometric methods were performed for the simultaneous analysis of metformin hydrochloride, empagliflozine, linagliptin, and pioglitazone hydrochloride in their synthetic mixtures without prior chemical separation. The drugs are approved for the treatment of type 2 diabetes. Despite the various advances in the management of diabetes, it remains to be the major cause of disability and morbidity, including blindness, amputation, heart disease, peripheral neuropathy, and kidney disease. These techniques consisted of several steps using ratio and derivative spectra. The absorption spectra of the mentioned drugs were recorded in the range of 200-350 nm, which have the concentration ranges of 1.0-10, 2.5-30, 5.0-40, and 2.5-30 µg mL-1 for metformin hydrochloride, empagliflozine, linagliptin, and pioglitazone hydrochloride, respectively, using zero-order spectra. The mean centring of ratio spectra combined with triple divisor were measured at the amplitude values 242, 256, 272 and 296 nm for metformin hydrochloride, empagliflozine, linagliptin and pioglitazone hydrochloride, respectively; the derivative ratio spectra-zero crossing quantifies the amplitude value of the analytical signal at 234, 244, 260 and 280 nm for metformin hydrochloride, empagliflozine, linagliptin and pioglitazone hydrochloride, respectively. The method was validated according to the ICH guidelines, accuracy, precision and repeatability were found to be within the acceptable limits. Finally, statistical comparisons between the proposed methods and with the reported methods with respect to accuracy and precision show that no significant difference was found by using Student's t-test, the F-test and one-way ANOVA.

A new stability indicating reverse phase high performance liquid chromatography method for the determination of enantiomeric purity of a DPP-4 inhibitor drug linagliptin.

A simple, sensitive, and stability indicating isocratic reverse phase high performance liquid chromatography method has been developed, optimized and validated for the separation and quantification of S-enantiomer in linagliptin (R-enantiomer) drug substance. Enantiomeric separation was achieved on a Cellulose tris(4-chloro-3-methylphenylcarbamate) stationary phase. Mobile phase consists of aqueous diammonium hydrogen phosphate buffer and acetonitrile in the ratio of 35:65 v/v. Isocratic elution was performed at a flow rate of 1.0 mL/min, the column oven temperature was set at 40°C and detection was at 226 nm. The resolution between R and S enantiomers is found to be more than 4.0. The impact of mobile phase composition, pH of buffer and temperature on the resolution has been studied. The detector response is found to be linear over the concentration range of 0.17-1.7 µg/mL. LOD and LOQ levels of S-enantiomer are found to be 0.057 and 0.172 µg/mL respectively. The recovery of S-enantiomer is 99.8% w/w. The proposed method is validated for specificity, precision, linearity, accuracy and robustness.

Dissolution or disintegration - substitution of dissolution by disintegration testing for a fixed dose combination product.

According to International Council for Harmonisation (ICH) guideline Q6A, dissolution testing can be replaced by disintegration testing if it can be shown that the active pharmaceutical ingredient is highly soluble and the formulation is rapidly releasing. In addition, a relationship between dissolution and disintegration has to be established. For a fixed-dose combination tablet of empagliflozin and linagliptin, this relationship was established by applying two different approaches. In the first approach, the extent to which the disintegration process of the film-coated tablets contributes to the release of the active ingredients was investigated. In the second approach, the mean disintegration times in a disintegration tester were correlated with the mean dissolution rates at a selected sampling time point. By correlating disintegration times in the dissolution vessel with the dissolution rate at selected sampling times it is demonstrated that the disintegration into primary particles is the rate limiting step for the dissolution process. A direct correlation of disintegration times in the disintegration tester with dissolution rate at a selected sampling time is established supporting a relationship between dissolution and disintegration testing for this type of formulation. Additionally, it could also be shown that the disintegration test method exhibits at least a similar discriminatory power compared to the proposed dissolution method. Based on a statistical approach and data from a bioavailability study, a clinical relevant specification for the disintegration time was established. All presented data support the replacement of dissolution by disintegration testing according to ICH Q6A for the selected fixed-dose combination product.

Simultaneous spectrophotometric determination of compounds having relatively disparate absorbance and concentration ranges; application to antidiabetic formulation of linagliptin and metformin.

The limited linear range of UV-Visible spectrophotometry may be insufficient to occupy multiple components with wide variations in their concentrations or absorptivities that will hinder the simultaneous spectrophotometric determination and may require spiking or measurements in subsequent dilution steps. The current work introduces the absorptivity target concentration (ATC) values, a simple way for the proper choice of the working spectral region to execute accurate and linear spectrophotometric measurements. Simultaneous spectrophotometric determination of linagliptin (LNG) and metformin (MET) that are present in a ratio of 1:400 was carried out using traditional spectrophotometric techniques such as third derivative and derivative ratio as well as recently developed techniques such as ratio difference and factorized dual wavelength. The proposed methods were able to determine MET in the concentration range of 50-1200 µg mL-1. On the other hand, LNG was successfully determined from its zero-order absorption UV-spectrum at λmax (296 nm) in the concentration range 2.5-25 µg mL-1. The mentioned methods were successfully applied for the determination of the LNG and MET in their combined dosage form. The methods were validated according to the ICH guidelines. The proposed ATC value can be employed as a novel concept for the proper choice of the working spectral region where spectrophotometric measurements can be deployed accurately and precisely.